106 results on '"Shih PM"'
Search Results
2. Expression of Dehydroshikimate Dehydratase in Sorghum Improves Biomass Yield, Accumulation of Protocatechuate, and Biorefinery Economics
- Author
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Tian, Y, Tian, Y, Yang, M, Lin, CY, Park, JH, Wu, CY, Kakumanu, R, De Ben, CM, Dalton, J, Vuu, KM, Shih, PM, Baidoo, EEK, Temple, S, Putnam, DH, Scheller, HV, Scown, CD, Eudes, A, Tian, Y, Tian, Y, Yang, M, Lin, CY, Park, JH, Wu, CY, Kakumanu, R, De Ben, CM, Dalton, J, Vuu, KM, Shih, PM, Baidoo, EEK, Temple, S, Putnam, DH, Scheller, HV, Scown, CD, and Eudes, A
- Abstract
Engineering bioenergy crops to accumulate value-added coproducts in planta is an attractive approach to increasing the value of lignocellulosic biomass and enabling a sustainable bioeconomy. In this study, we engineered sorghum with a bacterial gene encoding a dehydroshikimate dehydratase (qsuB) to convert the endogenous pool of 3-dehydroshikimate into the valuable compound protocatechuate (DHBA). We find that, when grown under field conditions, transgenic sorghum lines can accumulate up to 0.3% DHBA in stover on a dry weight (DW) basis without showing any difference in cell wall composition. An unexpected finding was an increase in yield for all qsuB-expressing lines. The grain yield and total biomass yield were 71 and 29% higher in the highest yielding line, respectively. On average, the total biomass yield of the engineered lines was 22.3 t/ha (DW). Moreover, we conducted a techno-economic analysis to investigate the economic impact of coproducing DHBA along with bioethanol in an integrated cellulosic biorefinery. Using engineered biomass sorghum with 0.3 DW% DHBA accumulated in planta as the feedstock, the economics of the integrated biorefineries has the potential to be improved. Our data demonstrate an engineering strategy to overproduce DHBA in bioenergy crops to facilitate sustainable manufacturing of biofuels and bioproducts.
- Published
- 2022
3. Agrobacterium tumefaciens: A Bacterium Primed for Synthetic Biology
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Thompson, MG, Thompson, MG, Moore, WM, Hummel, NFC, Pearson, AN, Barnum, CR, Scheller, HV, Shih, PM, Thompson, MG, Thompson, MG, Moore, WM, Hummel, NFC, Pearson, AN, Barnum, CR, Scheller, HV, and Shih, PM
- Abstract
Agrobacterium tumefaciens is an important tool in plant biotechnology due to its natural ability to transfer DNA into the genomes of host plants. Genetic manipulations of A. tumefaciens have yielded considerable advances in increasing transformational efficiency in a number of plant species and cultivars. Moreover, there is overwhelming evidence that modulating the expression of various mediators of A. tumefaciens virulence can lead to more successful plant transformation; thus, the application of synthetic biology to enable targeted engineering of the bacterium may enable new opportunities for advancing plant biotechnology. In this review, we highlight engineering targets in both A. tumefaciens and plant hosts that could be exploited more effectively through precision genetic control to generate high-quality transformation events in a wider range of host plants. We then further discuss the current state of A. tumefaciens and plant engineering with regard to plant transformation and describe how future work may incorporate a rigorous synthetic biology approach to tailor strains of A. tumefaciens used in plant transformation.
- Published
- 2020
4. Phototrophy and carbon fixation in Chlorobi postdate the rise of oxygen
- Author
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Ward, LM, primary and Shih, PM, additional
- Published
- 2021
- Full Text
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5. Author Correction: Hydrogen-based metabolism as an ancestral trait in lineages sibling to the Cyanobacteria.
- Author
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Matheus Carnevali, PB, Schulz, F, Castelle, CJ, Kantor, RS, Shih, PM, Sharon, I, Santini, JM, Olm, MR, Amano, Y, Thomas, BC, Anantharaman, K, Burstein, D, Becraft, ED, Stepanauskas, R, Woyke, T, Banfield, JF, Matheus Carnevali, PB, Schulz, F, Castelle, CJ, Kantor, RS, Shih, PM, Sharon, I, Santini, JM, Olm, MR, Amano, Y, Thomas, BC, Anantharaman, K, Burstein, D, Becraft, ED, Stepanauskas, R, Woyke, T, and Banfield, JF
- Abstract
The original version of this Article contained errors in Fig. 4. In panel a, the labels 'F420-reducing NiFe hydrogenase (group 3a)' and 'Group 2 NiFe hydrogenase' were misplaced. These errors have been corrected in both the PDF and HTML versions of the Article.
- Published
- 2019
6. Hydrogen-based metabolism as an ancestral trait in lineages sibling to the Cyanobacteria.
- Author
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Matheus Carnevali, PB, Schulz, F, Castelle, CJ, Kantor, RS, Shih, PM, Sharon, I, Santini, JM, Olm, MR, Amano, Y, Thomas, BC, Anantharaman, K, Burstein, D, Becraft, ED, Stepanauskas, R, Woyke, T, Banfield, JF, Matheus Carnevali, PB, Schulz, F, Castelle, CJ, Kantor, RS, Shih, PM, Sharon, I, Santini, JM, Olm, MR, Amano, Y, Thomas, BC, Anantharaman, K, Burstein, D, Becraft, ED, Stepanauskas, R, Woyke, T, and Banfield, JF
- Abstract
The evolution of aerobic respiration was likely linked to the origins of oxygenic Cyanobacteria. Close phylogenetic neighbors to Cyanobacteria, such as Margulisbacteria (RBX-1 and ZB3), Saganbacteria (WOR-1), Melainabacteria and Sericytochromatia, may constrain the metabolic platform in which aerobic respiration arose. Here, we analyze genomic sequences and predict that sediment-associated Margulisbacteria have a fermentation-based metabolism featuring a variety of hydrogenases, a streamlined nitrogenase, and electron bifurcating complexes involved in cycling of reducing equivalents. The genomes of ocean-associated Margulisbacteria encode an electron transport chain that may support aerobic growth. Some Saganbacteria genomes encode various hydrogenases, and others may be able to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Similarly, Melainabacteria have diverse energy metabolisms and are capable of fermentation and aerobic or anaerobic respiration. The ancestor of all these groups may have been an anaerobe in which fermentation and H2 metabolism were central metabolic features. The ability to use O2 as a terminal electron acceptor must have been subsequently acquired by these lineages.
- Published
- 2019
7. Phanerozoic Radiation of Ammonia Oxidizing Bacteria
- Author
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Ward, LM, primary, Johnston, DT, additional, and Shih, PM, additional
- Published
- 2019
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8. Gene stacking of multiple traits for high yield of fermentable sugars in plant biomass
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Aznar, A, Chalvin, C, Shih, PM, Maimann, M, Ebert, B, Birdseye, DS, Loque, D, Scheller, HV, Aznar, A, Chalvin, C, Shih, PM, Maimann, M, Ebert, B, Birdseye, DS, Loque, D, and Scheller, HV
- Abstract
BACKGROUND: Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production. RESULTS: We have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase and the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells. CONCLUSION: The results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.
- Published
- 2018
9. Engineered reduction of S-adenosylmethionine alters lignin in sorghum.
- Author
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Tian Y, Gao Y, Turumtay H, Turumtay EA, Chai YN, Choudhary H, Park JH, Wu CY, De Ben CM, Dalton J, Louie KB, Harwood T, Chin D, Vuu KM, Bowen BP, Shih PM, Baidoo EEK, Northen TR, Simmons BA, Hutmacher R, Atim J, Putnam DH, Scown CD, Mortimer JC, Scheller HV, and Eudes A
- Abstract
Background: Lignin is an aromatic polymer deposited in secondary cell walls of higher plants to provide strength, rigidity, and hydrophobicity to vascular tissues. Due to its interconnections with cell wall polysaccharides, lignin plays important roles during plant growth and defense, but also has a negative impact on industrial processes aimed at obtaining monosaccharides from plant biomass. Engineering lignin offers a solution to this issue. For example, previous work showed that heterologous expression of a coliphage S-adenosylmethionine hydrolase (AdoMetase) was an effective approach to reduce lignin in the model plant Arabidopsis. The efficacy of this engineering strategy remains to be evaluated in bioenergy crops., Results: We studied the impact of expressing AdoMetase on lignin synthesis in sorghum (Sorghum bicolor L. Moench). Lignin content, monomer composition, and size, as well as biomass saccharification efficiency were determined in transgenic sorghum lines. The transcriptome and metabolome were analyzed in stems at three developmental stages. Plant growth and biomass composition was further evaluated under field conditions. Results evidenced that lignin was reduced by 18% in the best transgenic line, presumably due to reduced activity of the S-adenosylmethionine-dependent O-methyltransferases involved in lignin synthesis. The modified sorghum features altered lignin monomer composition and increased lignin molecular weights. The degree of methylation of glucuronic acid on xylan was reduced. These changes enabled a ~20% increase in glucose yield after biomass pretreatment and saccharification compared to wild type. RNA-seq and untargeted metabolomic analyses evidenced some pleiotropic effects associated with AdoMetase expression. The transgenic sorghum showed developmental delay and reduced biomass yields at harvest, especially under field growing conditions., Conclusions: The expression of AdoMetase represents an effective lignin engineering approach in sorghum. However, considering that this strategy potentially impacts multiple S-adenosylmethionine-dependent methyltransferases, adequate promoters for fine-tuning AdoMetase expression will be needed to mitigate yield penalty., (© 2024. The Author(s).)
- Published
- 2024
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10. Design and Characterization of a Transcriptional Repression Toolkit for Plants.
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Markel K, Sabety J, Wijesinghe S, and Shih PM
- Abstract
Regulation of gene expression is essential for all life. Tools to manipulate the gene expression level have therefore proven to be very valuable in efforts to engineer biological systems. However, there are few well-characterized genetic parts that reduce gene expression in plants, commonly known as transcriptional repressors. We characterized the repression activity of a library consisting of repression motifs from approximately 25% of the members of the largest known family of repressors. Combining sequence information with our trans-regulatory function data, we next generated a library of synthetic transcriptional repression motifs with function predicted in advance. After characterizing our synthetic library, we demonstrated not only that many of our synthetic constructs were functional as repressors but also that our advance predictions of repression strength were better than random guesses. Finally, we assessed the functionality of known transcriptional repression motifs from a wide range of eukaryotes. Our study represents the largest plant repressor motif library experimentally characterized to date, providing unique opportunities for tuning transcription in plants.
- Published
- 2024
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11. Structural and biochemical basis for regiospecificity of the flavonoid glycosyltransferase UGT95A1.
- Author
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Sirirungruang S, Blay V, Scott YF, Pereira JH, Hammel M, Barnum CR, Adams PD, and Shih PM
- Subjects
- Glycosylation, Substrate Specificity, Flavonoids metabolism, Flavonoids chemistry, Crystallography, X-Ray, Plant Proteins metabolism, Plant Proteins chemistry, Plant Proteins genetics, Binding Sites, Luteolin chemistry, Luteolin metabolism, Models, Molecular, Protein Conformation, Glycosyltransferases metabolism, Glycosyltransferases chemistry, Glycosyltransferases genetics
- Abstract
Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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12. Evolution and origins of rubisco.
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Taylor-Kearney LJ, Wang RZ, and Shih PM
- Subjects
- Plants enzymology, Plants metabolism, Photosynthesis, Carbon Dioxide metabolism, Ribulose-Bisphosphate Carboxylase metabolism, Ribulose-Bisphosphate Carboxylase genetics, Evolution, Molecular
- Abstract
Rubisco (D-ribulose 1,5-bisphosphate carboxylase/oxygenase) is the most abundant enzyme in the world, constituting up to half of the soluble protein content in plant leaves. Such is its ubiquity that its chemical fingerprint can be detected in the geological record spanning billions of years. Rubisco catalyses the conversion of inorganic CO
2 into organic sugars, which underpin almost all of the biosphere, including our entire food chain. Due to its central role in the global carbon cycle, rubisco has been the subject of intense research for over 50 years. Rubisco is often considered inefficient due to its slow rate of carboxylation compared with other central metabolism enzymes, and its promiscuous oxygenase activity, which competes with the productive carboxylation reaction. It is hoped that engineering improved CO2 fixation will have significant advantages in agriculture and climate change mitigation. However, rubisco has proven difficult to engineer, with decades of efforts yielding limited results. Recent research has focused on reconstructing the evolutionary trajectory of rubisco to help elucidate its cryptic origins. Such evolutionary studies have led to a better understanding of both the origins of more complex rubisco forms and the broader relationship between rubisco's structure and function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)- Published
- 2024
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13. Systematic identification of transcriptional activation domains from non-transcription factor proteins in plants and yeast.
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Hummel NFC, Markel K, Stefani J, Staller MV, and Shih PM
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- Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Protein Domains genetics, Proteome metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Arabidopsis genetics, Arabidopsis metabolism, Transcriptional Activation genetics, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
Transcription factors can promote gene expression through activation domains. Whole-genome screens have systematically mapped activation domains in transcription factors but not in non-transcription factor proteins (e.g., chromatin regulators and coactivators). To fill this knowledge gap, we employed the activation domain predictor PADDLE to analyze the proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae. We screened 18,000 predicted activation domains from >800 non-transcription factor genes in both species, confirming that 89% of candidate proteins contain active fragments. Our work enables the annotation of hundreds of nuclear proteins as putative coactivators, many of which have never been ascribed any function in plants. Analysis of peptide sequence compositions reveals how the distribution of key amino acids dictates activity. Finally, we validated short, "universal" activation domains with comparable performance to state-of-the-art activation domains used for genome engineering. Our approach enables the genome-wide discovery and annotation of activation domains that can function across diverse eukaryotes., Competing Interests: Declaration of interests P.M.S. and N.F.C.H. have a patent pending for the library of synthetic TFs and all derived parts under US patent application number 63/579,836., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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14. Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides.
- Author
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Barnum CR, Paviani B, Couture G, Masarweh C, Chen Y, Huang YP, Markel K, Mills DA, Lebrilla CB, Barile D, Yang M, and Shih PM
- Subjects
- Humans, Prebiotics, Photosynthesis, Oligosaccharides metabolism, Milk, Human metabolism, Milk, Human chemistry, Plants, Genetically Modified genetics
- Abstract
Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs., (© 2024. The Author(s).)
- Published
- 2024
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15. Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls.
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Markel K, Novak V, Bowen BP, Tian Y, Chen YC, Sirirungruang S, Zhou A, Louie KB, Northen TR, Eudes A, Scheller HV, and Shih PM
- Subjects
- Animals, Quercus metabolism, Quercus parasitology, Plant Leaves metabolism, Plant Leaves parasitology, Lignin metabolism, Cell Wall metabolism, Wasps physiology, Plant Tumors parasitology, Host-Parasite Interactions
- Abstract
Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls, in which insect larva can develop while being sheltered and feeding on the plant. In particular, cynipid (Hymenoptera: Cynipidae) wasps have evolved to form morphologically complex galls and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is cell wall deposition that dictates the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how valley oak (Quercus lobata) leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form unique structures and organs., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)
- Published
- 2024
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16. A map of the rubisco biochemical landscape.
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Prywes N, Philips NR, Oltrogge LM, Lindner S, Candace Tsai YC, de Pins B, Cowan AE, Taylor-Kearney LJ, Chang HA, Hall LN, Bellieny-Rabelo D, Nisonoff HM, Weissman RF, Flamholz AI, Ding D, Bhatt AY, Shih PM, Mueller-Cajar O, Milo R, and Savage DF
- Abstract
Rubisco is the primary CO
2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.- Published
- 2024
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17. Engineering Brassica Crops to Optimize Delivery of Bioactive Products Postcooking.
- Author
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Barnum CR, Cho MJ, Markel K, and Shih PM
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- Humans, Glucosinolates analysis, Cooking, Crops, Agricultural genetics, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Isothiocyanates metabolism, Brassica genetics
- Abstract
Glucosinolates are plant-specialized metabolites that can be hydrolyzed by glycosyl hydrolases, called myrosinases, creating a variety of hydrolysis products that benefit human health. While cruciferous vegetables are a rich source of glucosinolates, they are often cooked before consumption, limiting the conversion of glucosinolates to hydrolysis products due to the denaturation of myrosinases. Here we screen a panel of glycosyl hydrolases for high thermostability and engineer the Brassica crop, broccoli ( Brassica oleracea L.), for the improved conversion of glucosinolates to chemopreventive hydrolysis products. Our transgenic broccoli lines enabled glucosinolate hydrolysis to occur at higher cooking temperatures, 20 °C higher than in wild-type broccoli. The process of cooking fundamentally transforms the bioavailability of many health-relevant bioactive compounds in our diet. Our findings demonstrate the promise of leveraging genetic engineering to tailor crops with novel traits that cannot be achieved through conventional breeding and improve the nutritional properties of the plants we consume.
- Published
- 2024
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18. Deep-branching evolutionary intermediates reveal structural origins of form I rubisco.
- Author
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Liu AK, Kaeser B, Chen L, West-Roberts J, Taylor-Kearney LJ, Lavy A, Günzing D, Li WJ, Hammel M, Nogales E, Banfield JF, and Shih PM
- Subjects
- Ribulose-Bisphosphate Carboxylase genetics, Ribulose-Bisphosphate Carboxylase chemistry, Ribulose-Bisphosphate Carboxylase metabolism
- Abstract
The enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the majority of biological carbon fixation on Earth. Although the vast majority of rubiscos across the tree of life assemble as homo-oligomers, the globally predominant form I enzyme-found in plants, algae, and cyanobacteria-forms a unique hetero-oligomeric complex. The recent discovery of a homo-oligomeric sister group to form I rubisco (named form I') has filled a key gap in our understanding of the enigmatic origins of the form I clade. However, to elucidate the series of molecular events leading to the evolution of form I rubisco, we must examine more distantly related sibling clades to contextualize the molecular features distinguishing form I and form I' rubiscos. Here, we present a comparative structural study retracing the evolutionary history of rubisco that reveals a complex structural trajectory leading to the ultimate hetero-oligomerization of the form I clade. We structurally characterize the oligomeric states of deep-branching form Iα and I'' rubiscos recently discovered from metagenomes, which represent key evolutionary intermediates preceding the form I clade. We further solve the structure of form I'' rubisco, revealing the molecular determinants that likely primed the enzyme core for the transition from a homo-oligomer to a hetero-oligomer. Our findings yield new insight into the evolutionary trajectory underpinning the adoption and entrenchment of the prevalent assembly of form I rubisco, providing additional context when viewing the enzyme family through the broader lens of protein evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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19. Harnessing Plant Sugar Metabolism for Glycoengineering.
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Tang SN, Barnum CR, Szarzanowicz MJ, Sirirungruang S, and Shih PM
- Abstract
Plants possess an innate ability to generate vast amounts of sugar and produce a range of sugar-derived compounds that can be utilized for applications in industry, health, and agriculture. Nucleotide sugars lie at the unique intersection of primary and specialized metabolism, enabling the biosynthesis of numerous molecules ranging from small glycosides to complex polysaccharides. Plants are tolerant to perturbations to their balance of nucleotide sugars, allowing for the overproduction of endogenous nucleotide sugars to push flux towards a particular product without necessitating the re-engineering of upstream pathways. Pathways to produce even non-native nucleotide sugars may be introduced to synthesize entirely novel products. Heterologously expressed glycosyltransferases capable of unique sugar chemistries can further widen the synthetic repertoire of a plant, and transporters can increase the amount of nucleotide sugars available to glycosyltransferases. In this opinion piece, we examine recent successes and potential future uses of engineered nucleotide sugar biosynthetic, transport, and utilization pathways to improve the production of target compounds. Additionally, we highlight current efforts to engineer glycosyltransferases. Ultimately, the robust nature of plant sugar biochemistry renders plants a powerful chassis for the production of target glycoconjugates and glycans.
- Published
- 2023
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20. Common origin of sterol biosynthesis points to a feeding strategy shift in Neoproterozoic animals.
- Author
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Brunoir T, Mulligan C, Sistiaga A, Vuu KM, Shih PM, O'Reilly SS, Summons RE, and Gold DA
- Subjects
- Animals, Sterols, Biological Evolution, Fossils, Phytosterols, Rhodophyta
- Abstract
Steranes preserved in sedimentary rocks serve as molecular fossils, which are thought to record the expansion of eukaryote life through the Neoproterozoic Era ( ~ 1000-541 Ma). Scientists hypothesize that ancient C
27 steranes originated from cholesterol, the major sterol produced by living red algae and animals. Similarly, C28 and C29 steranes are thought to be derived from the sterols of prehistoric fungi, green algae, and other microbial eukaryotes. However, recent work on annelid worms-an advanced group of eumetazoan animals-shows that they are also capable of producing C28 and C29 sterols. In this paper, we explore the evolutionary history of the 24-C sterol methyltransferase (smt) gene in animals, which is required to make C28+ sterols. We find evidence that the smt gene was vertically inherited through animals, suggesting early eumetazoans were capable of C28+ sterol synthesis. Our molecular clock of the animal smt gene demonstrates that its diversification coincides with the rise of C28 and C29 steranes in the Neoproterozoic. This study supports the hypothesis that early eumetazoans were capable of making C28+ sterols and that many animal lineages independently abandoned its biosynthesis around the end-Neoproterozoic, coinciding with the rise of abundant eukaryotic prey., (© 2023. The Author(s).)- Published
- 2023
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21. Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials.
- Author
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Buell CR, Dardick C, Parrott W, Schmitz RJ, Shih PM, Tsai CJ, and Urbanowicz B
- Abstract
Humans have been modifying plant traits for thousands of years, first through selection (i.e., domestication) then modern breeding, and in the last 30 years, through biotechnology. These modifications have resulted in increased yield, more efficient agronomic practices, and enhanced quality traits. Precision knowledge of gene regulation and function through high-resolution single-cell omics technologies, coupled with the ability to engineer plant genomes at the DNA sequence, chromatin accessibility, and gene expression levels, can enable engineering of complex and complementary traits at the biosystem level. Populus spp., the primary genetic model system for woody perennials, are among the fastest growing trees in temperate zones and are important for both carbon sequestration and global carbon cycling. Ample genomic and transcriptomic resources for poplar are available including emerging single-cell omics datasets. To expand use of poplar outside of valorization of woody biomass, chassis with novel morphotypes in which stem branching and tree height are modified can be fabricated thereby leading to trees with altered leaf to wood ratios. These morphotypes can then be engineered into customized chemotypes that produce high value biofuels, bioproducts, and biomaterials not only in specific organs but also in a cell-type-specific manner. For example, the recent discovery of triterpene production in poplar leaf trichomes can be exploited using cell-type specific regulatory sequences to synthesize high value terpenes such as the jet fuel precursor bisabolene specifically in the trichomes. By spatially and temporally controlling expression, not only can pools of abundant precursors be exploited but engineered molecules can be sequestered in discrete cell structures in the leaf. The structural diversity of the hemicellulose xylan is a barrier to fully utilizing lignocellulose in biomaterial production and by leveraging cell-type-specific omics data, cell wall composition can be modified in a tailored and targeted specific manner to generate poplar wood with novel chemical features that are amenable for processing or advanced manufacturing. Precision engineering poplar as a multi-purpose sustainable feedstock highlights how genome engineering can be used to re-imagine a crop species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Buell, Dardick, Parrott, Schmitz, Shih, Tsai and Urbanowicz.)
- Published
- 2023
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22. Plant-based production of diverse human milk oligosaccharides.
- Author
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Barnum CR, Paviani B, Couture G, Masarweh C, Chen Y, Huang YP, Mills DA, Lebrilla CB, Barile D, Yang M, and Shih PM
- Abstract
Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates that aid in the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the ∼130 structurally diverse HMOs at scale has proven difficult. Here, we produce a vast diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high value HMOs, such as lacto-N-fucopentaose I, that have not yet been commercially produced using state-of-the-art microbial fermentative processes. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the cheap and sustainable production of HMOs.
- Published
- 2023
- Full Text
- View/download PDF
23. Systematic identification of transcriptional activator domains from non-transcription factor proteins in plants and yeast.
- Author
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Hummel NFC, Markel K, Stefani J, Staller MV, and Shih PM
- Abstract
Transcription factors promote gene expression via trans-regulatory activation domains. Although whole genome scale screens in model organisms (e.g. human, yeast, fly) have helped identify activation domains from transcription factors, such screens have been less extensively used to explore the occurrence of activation domains in non-transcription factor proteins, such as transcriptional coactivators, chromatin regulators and some cytosolic proteins, leaving a blind spot on what role activation domains in these proteins could play in regulating transcription. We utilized the activation domain predictor PADDLE to mine the entire proteomes of two model eukaryotes, Arabidopsis thaliana and Saccharomyces cerevisiae ( 1 ). We characterized 18,000 fragments covering predicted activation domains from >800 non-transcription factor genes in both species, and experimentally validated that 89% of proteins contained fragments capable of activating transcription in yeast. Peptides with similar sequence composition show a broad range of activities, which is explained by the arrangement of key amino acids. We also annotated hundreds of nuclear proteins with activation domains as putative coactivators; many of which have never been ascribed any function in plants. Furthermore, our library contains >250 non-nuclear proteins containing peptides with activation domain function across both eukaryotic lineages, suggesting that there are unknown biological roles of these peptides beyond transcription. Finally, we identify and validate short, 'universal' eukaryotic activation domains that activate transcription in both yeast and plants with comparable or stronger performance to state-of-the-art activation domains. Overall, our dual host screen provides a blueprint on how to systematically discover novel genetic parts for synthetic biology that function across a wide diversity of eukaryotes., Significance Statement: Activation domains promote transcription and play a critical role in regulating gene expression. Although the mapping of activation domains from transcription factors has been carried out in previous genome-wide screens, their occurrence in non-transcription factors has been less explored. We utilize an activation domain predictor to mine the entire proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae for new activation domains on non-transcription factor proteins. We validate peptides derived from >750 non-transcription factor proteins capable of activating transcription, discovering many potentially new coactivators in plants. Importantly, we identify novel genetic parts that can function across both species, representing unique synthetic biology tools.
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- 2023
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24. Transcriptome architecture of the three main lineages of agrobacteria.
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Waldburger L, Thompson MG, Weisberg AJ, Lee N, Chang JH, Keasling JD, and Shih PM
- Subjects
- Promoter Regions, Genetic, Gene Expression Regulation, Nucleotides, Transcriptome, Genomics
- Abstract
Agrobacteria are a diverse, polyphyletic group of prokaryotes with multipartite genomes capable of transferring DNA into the genomes of host plants, making them an essential tool in plant biotechnology. Despite their utility in plant transformation, genome-wide transcriptional regulation is not well understood across the three main lineages of agrobacteria. Transcription start sites (TSSs) are a necessary component of gene expression and regulation. In this study, we used differential RNA-seq and a TSS identification algorithm optimized on manually annotated TSS, then validated with existing TSS to identify thousands of TSS with nucleotide resolution for representatives of each lineage. We extend upon the 356 TSSs previously reported in Agrobacterium fabrum C58 by identifying 1,916 TSSs. In addition, we completed genomes and phenotyping of Rhizobium rhizogenes C16/80 and Allorhizobium vitis T60/94, identifying 2,650 and 2,432 TSSs, respectively. Parameter optimization was crucial for an accurate, high-resolution view of genome and transcriptional dynamics, highlighting the importance of algorithm optimization in genome-wide TSS identification and genomics at large. The optimized algorithm reduced the number of TSSs identified internal and antisense to the coding sequence on average by 90.5% and 91.9%, respectively. Comparison of TSS conservation between orthologs of the three lineages revealed differences in cell cycle regulation of ctrA as well as divergence of transcriptional regulation of chemotaxis-related genes when grown in conditions that simulate the plant environment. These results provide a framework to elucidate the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. IMPORTANCE Transcription start sites (TSSs) are fundamental for understanding gene expression and regulation. Agrobacteria, a group of prokaryotes with the ability to transfer DNA into the genomes of host plants, are widely used in plant biotechnology. However, the genome-wide transcriptional regulation of agrobacteria is not well understood, especially in less-studied lineages. Differential RNA-seq and an optimized algorithm enabled identification of thousands of TSSs with nucleotide resolution for representatives of each lineage. The results of this study provide a framework for elucidating the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. The optimized algorithm also highlights the importance of parameter optimization in genome-wide TSS identification and genomics at large., Competing Interests: J.D.K. has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms.
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- 2023
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25. Plant glycosyltransferases for expanding bioactive glycoside diversity.
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Sirirungruang S, Barnum CR, Tang SN, and Shih PM
- Subjects
- Glycosylation, Plants metabolism, Drug Discovery, Glycosyltransferases chemistry, Glycosides pharmacology, Glycosides chemistry
- Abstract
Glycosylation is a successful strategy to alter the pharmacological properties of small molecules, and it has emerged as a unique approach to expand the chemical space of natural products that can be explored in drug discovery. Traditionally, most glycosylation events have been carried out chemically, often requiring many protection and deprotection steps to achieve a target molecule. Enzymatic glycosylation by glycosyltransferases could provide an alternative strategy for producing new glycosides. In particular, the glycosyltransferase family has greatly expanded in plants, representing a rich enzymatic resource to mine and expand the diversity of glycosides with novel bioactive properties. This article highlights previous and prospective uses for plant glycosyltransferases in generating bioactive glycosides and altering their pharmacological properties.
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- 2023
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26. Alternative Identification of Glycosides Using MS/MS Matching with an In Silico-Modified Aglycone Mass Spectra Library.
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Rodríguez EP, Li Y, Vaniya A, Shih PM, and Fiehn O
- Subjects
- Chromatography, Liquid methods, Tissue Distribution, Spectrometry, Mass, Electrospray Ionization methods, Ions, Sugars, Chromatography, High Pressure Liquid methods, Glycosides analysis, Tandem Mass Spectrometry methods
- Abstract
Glycosylation of metabolites serves multiple purposes. Adding sugars makes metabolites more water soluble and improves their biodistribution, stability, and detoxification. In plants, the increase in melting points enables storing otherwise volatile compounds that are released by hydrolysis when needed. Classically, glycosylated metabolites were identified by mass spectrometry (MS/MS) using [M-sugar] neutral losses. Herein, we studied 71 pairs of glycosides with their respective aglycones, including hexose, pentose, and glucuronide moieties. Using liquid chromatography (LC) coupled to electrospray ionization high-resolution mass spectrometry, we detected the classic [M-sugar] product ions for only 68% of glycosides. Instead, we found that most aglycone MS/MS product ions were conserved in the MS/MS spectra of their corresponding glycosides, even when no [M-sugar] neutral losses were observed. We added pentose and hexose units to the precursor masses of an MS/MS library of 3057 aglycones to enable rapid identification of glycosylated natural products with standard MS/MS search algorithms. When searching unknown compounds in untargeted LC-MS/MS metabolomics data of chocolate and tea, we structurally annotated 108 novel glycosides in standard MS-DIAL data processing. We uploaded this new in silico-glycosylated product MS/MS library to GitHub to enable users to detect natural product glycosides without authentic chemical standards.
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- 2023
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27. How to engineer the unknown: Advancing a quantitative and predictive understanding of plant and soil biology to address climate change.
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Alamos S and Shih PM
- Subjects
- Plants genetics, Synthetic Biology, Photosynthesis genetics, Climate Change, Soil
- Abstract
Our basic understanding of carbon cycling in the biosphere remains qualitative and incomplete, precluding our ability to effectively engineer novel solutions to climate change. How can we attempt to engineer the unknown? This challenge has been faced before in plant biology, providing a roadmap to guide future efforts. We use examples from over a century of photosynthesis research to illustrate the key principles that will set future plant engineering on a solid footing, namely, an effort to identify the key control variables, quantify the effects of systematically tuning these variables, and use theory to account for these observations. The main contributions of plant synthetic biology will stem not from delivering desired genotypes but from enabling the kind of predictive understanding necessary to rationally design these genotypes in the first place. Only then will synthetic plant biology be able to live up to its promise., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Alamos, Shih. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2023
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28. The trans-regulatory landscape of gene networks in plants.
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Hummel NFC, Zhou A, Li B, Markel K, Ornelas IJ, and Shih PM
- Subjects
- Gene Regulatory Networks genetics, Transcription Factors genetics, Transcription Factors metabolism, Arabidopsis genetics, Arabidopsis Proteins genetics
- Abstract
The transcriptional effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative transcriptional effector domains of over 400 Arabidopsis thaliana transcription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how our characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems., Competing Interests: Declaration of interests P.M.S. and N.F.C.H. have a patent pending for the library of synthetic TFs and all derived parts under US patent application number 18/298,942., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2023
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29. The pGinger Family of Expression Plasmids.
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Pearson AN, Thompson MG, Kirkpatrick LD, Ho C, Vuu KM, Waldburger LM, Keasling JD, and Shih PM
- Subjects
- Plasmids genetics, Promoter Regions, Genetic, Escherichia coli genetics, Escherichia coli metabolism, Metabolic Engineering
- Abstract
The pGinger suite of expression plasmids comprises 43 plasmids that will enable precise constitutive and inducible gene expression in a wide range of Gram-negative bacterial species. Constitutive vectors are composed of 16 synthetic constitutive promoters upstream of red fluorescent protein (RFP), with a broad-host-range BBR1 origin and a kanamycin resistance marker. The family also has seven inducible systems (Jungle Express, Psal/NahR, Pm/XylS, Prha/RhaS, LacO1/LacI, LacUV5/LacI, and Ptet/TetR) controlling RFP expression on BBR1/kanamycin plasmid backbones. For four of these inducible systems (Jungle Express, Psal/NahR, LacO1/LacI, and Ptet/TetR), we created variants that utilize the RK2 origin and spectinomycin or gentamicin selection. Relevant RFP expression and growth data have been collected in the model bacterium Escherichia coli as well as Pseudomonas putida. All pGinger vectors are available via the Joint BioEnergy Institute (JBEI) Public Registry. IMPORTANCE Metabolic engineering and synthetic biology are predicated on the precise control of gene expression. As synthetic biology expands beyond model organisms, more tools will be required that function robustly in a wide range of bacterial hosts. The pGinger family of plasmids constitutes 43 plasmids that will enable both constitutive and inducible gene expression in a wide range of nonmodel Proteobacteria ., Competing Interests: The authors declare a conflict of interest. J.D.K. has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms.
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- 2023
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30. A dolabralexin-deficient mutant provides insight into specialized diterpenoid metabolism in maize.
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Murphy KM, Dowd T, Khalil A, Char SN, Yang B, Endelman BJ, Shih PM, Topp C, Schmelz EA, and Zerbe P
- Subjects
- Biosynthetic Pathways, Lipid Metabolism, Zea mays metabolism, Diterpenes metabolism
- Abstract
Two major groups of specialized metabolites in maize (Zea mays), termed kauralexins and dolabralexins, serve as known or predicted diterpenoid defenses against pathogens, herbivores, and other environmental stressors. To consider the physiological roles of the recently discovered dolabralexin pathway, we examined dolabralexin structural diversity, tissue-specificity, and stress-elicited production in a defined biosynthetic pathway mutant. Metabolomics analyses support a larger number of dolabralexin pathway products than previously known. We identified dolabradienol as a previously undetected pathway metabolite and characterized its enzymatic production. Transcript and metabolite profiling showed that dolabralexin biosynthesis and accumulation predominantly occur in primary roots and show quantitative variation across genetically diverse inbred lines. Generation and analysis of CRISPR-Cas9-derived loss-of-function Kaurene Synthase-Like 4 (Zmksl4) mutants demonstrated dolabralexin production deficiency, thus supporting ZmKSL4 as the diterpene synthase responsible for the conversion of geranylgeranyl pyrophosphate precursors into dolabradiene and downstream pathway products. Zmksl4 mutants further display altered root-to-shoot ratios and root architecture in response to water deficit. Collectively, these results demonstrate dolabralexin biosynthesis via ZmKSL4 as a committed pathway node biochemically separating kauralexin and dolabralexin metabolism, and suggest an interactive role of maize dolabralexins in plant vigor during abiotic stress., Competing Interests: Conflict of interest statement. The authors declare no competing interests in accordance with the journal policies., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2023
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31. A Suite of Constitutive Promoters for Tuning Gene Expression in Plants.
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Zhou A, Kirkpatrick LD, Ornelas IJ, Washington LJ, Hummel NFC, Gee CW, Tang SN, Barnum CR, Scheller HV, and Shih PM
- Subjects
- Promoter Regions, Genetic genetics, Transgenes genetics, Plasmids genetics, Gene Expression, Plants genetics
- Abstract
The need for convenient tools to express transgenes over a large dynamic range is pervasive throughout plant synthetic biology; however, current efforts are largely limited by the heavy reliance on a small set of strong promoters, precluding more nuanced and refined engineering endeavors in planta . To address this technical gap, we characterize a suite of constitutive promoters that span a wide range of transcriptional levels and develop a GoldenGate-based plasmid toolkit named PCONS, optimized for versatile cloning and rapid testing of transgene expression at varying strengths. We demonstrate how easy access to a stepwise gradient of expression levels can be used for optimizing synthetic transcriptional systems and the production of small molecules in planta . We also systematically investigate the potential of using PCONS as an internal standard in plant biology experimental design, establishing the best practices for signal normalization in experiments. Although our library has primarily been developed for optimizing expression in N. benthamiana , we demonstrate the translatability of our promoters across distantly related species using a multiplexed reporter assay with barcoded transcripts. Our findings showcase the advantages of the PCONS library as an invaluable toolkit for plant synthetic biology.
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- 2023
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32. Carbon isotope fractionation by an ancestral rubisco suggests that biological proxies for CO 2 through geologic time should be reevaluated.
- Author
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Wang RZ, Nichols RJ, Liu AK, Flamholz AI, Artier J, Banda DM, Savage DF, Eiler JM, Shih PM, and Fischer WW
- Subjects
- Carbon Isotopes metabolism, Carbon metabolism, Photosynthesis, Ribulose-Bisphosphate Carboxylase metabolism, Carbon Dioxide metabolism
- Abstract
The history of Earth's carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO
2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. Therefore, we measured both biomass (εp ) and enzymatic (εRubisco ) carbon isotope fractionations of a cyanobacterial strain ( Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger εp values than WT, despite having a much smaller εRubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC εp exceeded ANC εRubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO2 from geological data. Understanding the evolution of rubisco and the CO2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2 .- Published
- 2023
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33. A Bacterial Form I' Rubisco Has a Smaller Carbon Isotope Fractionation than Its Form I Counterpart.
- Author
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Wang RZ, Liu AK, Banda DM, Fischer WW, and Shih PM
- Subjects
- Carbon Isotopes, Ribulose-Bisphosphate Carboxylase metabolism, Bacterial Proteins metabolism
- Abstract
Form I rubiscos evolved in Cyanobacteria ≥ 2.5 billion years ago and are enzymatically unique due to the presence of small subunits (RbcS) capping both ends of an octameric large subunit (RbcL) rubisco assembly to form a hexadecameric (L
8 S8 ) holoenzyme. Although RbcS was previously thought to be integral to Form I rubisco stability, the recent discovery of a closely related sister clade of octameric rubiscos (Form I'; L8 ) demonstrates that the L8 complex can assemble without small subunits (Banda et al. 2020). Rubisco also displays a kinetic isotope effect (KIE) where the 3PG product is depleted in13 C relative to12 C. In Cyanobacteria, only two Form I KIE measurements exist, making interpretation of bacterial carbon isotope data difficult. To aid comparison, we measured in vitro the KIEs of Form I' ( Candidatus Promineofilum breve) and Form I ( Synechococcus elongatus PCC 6301) rubiscos and found the KIE to be smaller in the L8 rubisco (16.25 ± 1.36‱ vs. 22.42 ± 2.37‱, respectively). Therefore, while small subunits may not be necessary for protein stability, they may affect the KIE. Our findings may provide insight into the function of RbcS and allow more refined interpretation of environmental carbon isotope data.- Published
- 2023
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34. Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production.
- Author
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Valencia LE, Incha MR, Schmidt M, Pearson AN, Thompson MG, Roberts JB, Mehling M, Yin K, Sun N, Oka A, Shih PM, Blank LM, Gladden J, and Keasling JD
- Subjects
- Fatty Acids, Nonesterified metabolism, Biofuels, Biomass, Fatty Acids metabolism, Pseudomonas putida genetics, Pseudomonas putida metabolism
- Abstract
Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L. This work paves the road to produce high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host., (© 2022. The Author(s).)
- Published
- 2022
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35. Correction to "Expression of Dehydroshikimate Dehydratase in Sorghum Improves Biomass Yield, Accumulation of Protocatechuate, and Biorefinery Economics".
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Tian Y, Yang M, Lin CY, Park JH, Wu CY, Kakumanu R, De Ben CM, Dalton J, Vuu KM, Shih PM, Baidoo EEK, Temple S, Putnam DH, Scheller HV, Scown CD, and Eudes A
- Abstract
[This corrects the article DOI: 10.1021/acssuschemeng.2c01160.]., (© 2022 The Authors. Published by American Chemical Society.)
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- 2022
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36. Structural plasticity enables evolution and innovation of RuBisCO assemblies.
- Author
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Liu AK, Pereira JH, Kehl AJ, Rosenberg DJ, Orr DJ, Chu SKS, Banda DM, Hammel M, Adams PD, Siegel JB, and Shih PM
- Abstract
Oligomerization is a core structural feature that defines the form and function of many proteins. Most proteins form molecular complexes; however, there remains a dearth of diversity-driven structural studies investigating the evolutionary trajectory of these assemblies. Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is one such enzyme that adopts multiple assemblies, although the origins and distribution of its different oligomeric states remain cryptic. Here, we retrace the evolution of ancestral and extant form II RuBisCOs, revealing a complex and diverse history of oligomerization. We structurally characterize a newly discovered tetrameric RuBisCO, elucidating how solvent-exposed surfaces can readily adopt new interactions to interconvert or give rise to new oligomeric states. We further use these principles to engineer and demonstrate how changes in oligomerization can be mediated by relatively few mutations. Our findings yield insight into how structural plasticity may give rise to new oligomeric states.
- Published
- 2022
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37. Synthetic gene circuits take root.
- Author
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Alamos S and Shih PM
- Subjects
- Gene Expression Regulation, Plant, Gene Regulatory Networks, Genes, Synthetic, Plant Roots genetics, Plant Roots growth & development
- Abstract
Complex spatial patterns of gene expression are engineered in plants to modulate root morphology.
- Published
- 2022
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38. Phototrophy and carbon fixation in Chlorobi postdate the rise of oxygen.
- Author
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Ward LM and Shih PM
- Subjects
- Carbon Cycle, Iron metabolism, Oxygen metabolism, Photosynthesis, Phototrophic Processes, Phylogeny, Chlorobi genetics
- Abstract
While most productivity on the surface of the Earth today is fueled by oxygenic photosynthesis, for much of Earth history it is thought that anoxygenic photosynthesis-using compounds like ferrous iron or sulfide as electron donors-drove most global carbon fixation. Anoxygenic photosynthesis is still performed by diverse bacteria in niche environments today. Of these, the Chlorobi (formerly green sulfur bacteria) are often interpreted as being particularly ancient and are frequently proposed to have fueled the biosphere during late Archean and early Paleoproterozoic time before the rise of oxygenic photosynthesis. Here, we perform comparative genomic, phylogenetic, and molecular clock analyses to determine the antiquity of the Chlorobi and their characteristic phenotypes. We show that contrary to common assumptions, the Chlorobi clade is relatively young, with anoxygenic phototrophy, carbon fixation via the rTCA pathway, and iron oxidation all significantly postdating the rise of oxygen ~2.3 billion years ago. The Chlorobi therefore could not have fueled the Archean biosphere, but instead represent a relatively young radiation of organisms which likely acquired the capacity for anoxygenic photosynthesis and other traits via horizontal gene transfer sometime after the evolution of oxygenic Cyanobacteria., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2022
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39. Plant-based engineering for production of high-valued natural products.
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Sirirungruang S, Markel K, and Shih PM
- Subjects
- Agriculture, Humans, Plants genetics, Plants metabolism, Biological Products metabolism
- Abstract
Covering: up to March 2022Plants are a unique source of complex specialized metabolites, many of which play significant roles in human society. In many cases, however, the availability of these metabolites from naturally occurring sources fails to meet current demands. Thus, there is much interest in expanding the production capacity of target plant molecules. Traditionally, plant breeding, chemical synthesis, and microbial fermentation are considered the primary routes towards large scale production of natural products. Here, we explore the advances, challenges, and future of plant engineering as a complementary path. Although plants are an integral part of our food and agricultural systems and sustain an extensive array of chemical constituents, their complex genetics and physiology have prevented the optimal exploitation of plants as a production chassis. We highlight emerging engineering tools and scientific advances developed in recent years that have improved the prospects of using plants as a sustainable and scalable production platform. We also discuss technological limitations and overall economic outlook of plant-based production of natural products.
- Published
- 2022
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40. Optimization of Heterologous Glucoraphanin Production In Planta .
- Author
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Barnum CR, Endelman BJ, Ornelas IJ, Pignolet RM, and Shih PM
- Subjects
- Oximes, Sulfoxides, Nicotiana genetics, Glucosinolates, Imidoesters
- Abstract
Glucoraphanin is a plant specialized metabolite found in cruciferous vegetables that has long been a target for production in a heterologous host because it can subsequently be hydrolyzed to form the chemopreventive compound sulforaphane before and during consumption. However, previous studies have only been able to produce small amounts of glucoraphanin in heterologous plant and microbial systems compared to the levels found in glucoraphanin-producing plants, suggesting that there may be missing auxiliary genes that play a role in improving production in planta . In an effort to identify auxiliary genes required for high glucoraphanin production, we leveraged transient expression in Nicotiana benthamiana to screen a combination of previously uncharacterized coexpressed genes and rationally selected genes alongside the glucoraphanin biosynthetic pathway. This strategy alleviated metabolic bottlenecks, which improved glucoraphanin production by 4.74-fold. Our optimized glucoraphanin biosynthetic pathway provides a pathway amenable for high glucoraphanin production.
- Published
- 2022
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41. Heinz-resistant tomato cultivars exhibit a lignin-based resistance to field dodder (Cuscuta campestris) parasitism.
- Author
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Jhu MY, Farhi M, Wang L, Philbrook RN, Belcher MS, Nakayama H, Zumstein KS, Rowland SD, Ron M, Shih PM, and Sinha NR
- Subjects
- Host Specificity, Lignin, Cuscuta physiology, Solanum lycopersicum genetics, Solanum
- Abstract
Cuscuta species (dodders) are agriculturally destructive, parasitic angiosperms. These parasitic plants use haustoria as physiological bridges to extract nutrients and water from hosts. Cuscuta campestris has a broad host range and wide geographical distribution. While some wild tomato relatives are resistant, cultivated tomatoes are generally susceptible to C. campestris infestations. However, some specific Heinz tomato (Solanum lycopersicum) hybrid cultivars exhibit resistance to dodders in the field, but their defense mechanism was previously unknown. Here, we discovered that the stem cortex in these resistant lines responds with local lignification upon C. campestris attachment, preventing parasite entry into the host. Lignin Induction Factor 1 (LIF1, an AP2-like transcription factor), SlMYB55, and Cuscuta R-gene for Lignin-based Resistance 1, a CC-NBS-LRR (CuRLR1) are identified as factors that confer host resistance by regulating lignification. SlWRKY16 is upregulated upon C. campestris infestation and potentially negatively regulates LIF1 function. Intriguingly, CuRLR1 may play a role in signaling or function as an intracellular receptor for receiving Cuscuta signals or effectors, thereby regulating lignification-based resistance. In summary, these four regulators control the lignin-based resistance response in specific Heinz tomato cultivars, preventing C. campestris from parasitizing resistant tomatoes. This discovery provides a foundation for investigating multilayer resistance against Cuscuta species and has potential for application in other essential crops attacked by parasitic plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)
- Published
- 2022
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42. Nitrogen Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing.
- Author
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Schmidt M, Pearson AN, Incha MR, Thompson MG, Baidoo EEK, Kakumanu R, Mukhopadhyay A, Shih PM, Deutschbauer AM, Blank LM, and Keasling JD
- Subjects
- Amino Acids metabolism, Nitrogen metabolism, Phenotype, Transaminases genetics, Transaminases metabolism, Pseudomonas putida metabolism
- Abstract
Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop-out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro . Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCE Understanding the genetic basis of P. putida's diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators, which are of particular interest for metabolic engineering.
- Published
- 2022
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43. Overexpression of the rice BAHD acyltransferase AT10 increases xylan-bound p-coumarate and reduces lignin in Sorghum bicolor.
- Author
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Tian Y, Lin CY, Park JH, Wu CY, Kakumanu R, Pidatala VR, Vuu KM, Rodriguez A, Shih PM, Baidoo EEK, Temple S, Simmons BA, Gladden JM, Scheller HV, and Eudes A
- Abstract
Background: The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored., Results: We obtained eight independent sorghum (Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p-coumarate (pCA), a higher amount of p-coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of pCA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification., Conclusions: Expression of OsAT10 in sorghum leads to an increase of xylan-bound pCA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall pCA remains unchanged indicating that most pCA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound pCA and lignification in cell walls., (© 2021. The Author(s).)
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- 2021
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44. Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii.
- Author
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Wakao S, Shih PM, Guan K, Schackwitz W, Ye J, Patel D, Shih RM, Dent RM, Chovatia M, Sharma A, Martin J, Wei CL, and Niyogi KK
- Subjects
- Chlamydomonas reinhardtii metabolism, Gene Deletion, Gene Duplication, Acetates metabolism, Chlamydomonas reinhardtii genetics, Mutation, Photosynthesis genetics, Exome Sequencing
- Abstract
Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis., Competing Interests: The authors have declared that no competing interests exists.
- Published
- 2021
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45. Utilizing Plant Synthetic Biology to Improve Human Health and Wellness.
- Author
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Barnum CR, Endelman BJ, and Shih PM
- Abstract
Plants offer a vast source of bioactive chemicals with the potential to improve human health through the prevention and treatment of disease. However, many potential therapeutics are produced in small amounts or in species that are difficult to cultivate. The rapidly evolving field of plant synthetic biology provides tools to capitalize on the inventive chemistry of plants by transferring metabolic pathways for therapeutics into far more tenable plants, increasing our ability to produce complex pharmaceuticals in well-studied plant systems. Plant synthetic biology also provides methods to enhance the ability to fortify crops with nutrients and nutraceuticals. In this review, we discuss (1) the potential of plant synthetic biology to improve human health by generating plants that produce pharmaceuticals, nutrients, and nutraceuticals and (2) the technological challenges hindering our ability to generate plants producing health-promoting small molecules., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Barnum, Endelman and Shih.)
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- 2021
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46. From breeding to genome design: A genomic makeover for potatoes.
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Markel K and Shih PM
- Subjects
- Genome, Plant, Genomics, Phenotype, Plant Breeding, Solanum tuberosum genetics
- Abstract
Potato breeding efforts have long been hindered by the genetic consequences of millennia of clonal propagation. To mitigate genomic constraints, Zhang et al. leverage an unprecedented scale of sequencing and marker-assisted breeding to unlock traits that have not been possible through classical breeding, providing a blueprint for plant genome design., (Copyright © 2021 Elsevier Inc. All rights reserved.)
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- 2021
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47. CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants.
- Author
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Pan C, Wu X, Markel K, Malzahn AA, Kundagrami N, Sretenovic S, Zhang Y, Cheng Y, Shih PM, and Qi Y
- Subjects
- Crops, Agricultural genetics, Gene Expression Regulation, Plant, Genetic Variation, Genotype, Arabidopsis genetics, CRISPR-Cas Systems, Genetic Engineering methods, Solanum lycopersicum genetics, Oryza genetics, Plant Breeding methods, Transcriptional Activation genetics
- Abstract
RNA-guided CRISPR activation (CRISPRa) systems have been developed in plants. However, the simultaneous activation of multiple genes remains challenging. Here, we develop a highly robust CRISPRa system working in rice, Arabidopsis and tomato, CRISPR-Act3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenes Cas9 (dSpCas9). The CRISPR-Act3.0 system results in fourfold to sixfold higher activation than the state-of-the-art CRISPRa systems. We further develop a tRNA-gR2.0 (single guide RNA 2.0) expression system enabling CRISPR-Act3.0-based robust activation of up to seven genes for metabolic engineering in rice. In addition, CRISPR-Act3.0 allows the simultaneous modification of multiple traits in Arabidopsis, which are stably transmitted to the T3 generations. On the basis of CRISPR-Act3.0, we elucidate guide RNA targeting rules for effective transcriptional activation. To target T-rich protospacer adjacent motifs (PAMs), we transfer this activation strategy to CRISPR-dCas12b and further improve the dAaCas12b-based CRISPRa system. Moreover, we develop a potent near-PAM-less CRISPR-Act3.0 system on the basis of the SpRY dCas9 variant, which outperforms the dCas9-NG system in both activation potency and targeting scope. Altogether, our study has substantially improved the CRISPRa technology in plants and provided plant researchers a powerful toolbox for efficient gene activation in foundational and translational research., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2021
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48. In-planta production of the biodegradable polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC): Stacking reduced biomass recalcitrance with value-added co-product.
- Author
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Lin CY, Vuu KM, Amer B, Shih PM, Baidoo EEK, Scheller HV, and Eudes A
- Subjects
- Biomass, Lignin, Pyrones, Arabidopsis genetics, Polyesters
- Abstract
2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable intermediate that naturally occurs during microbial degradation of lignin by bacteria, represents a promising building block for diverse biomaterials and polyesters such as biodegradable plastics. The lack of a chemical synthesis method has hindered large-scale utilization of PDC and metabolic engineering approaches for its biosynthesis have recently emerged. In this study, we demonstrate a strategy for the production of PDC via manipulation of the shikimate pathway using plants as green factories. In tobacco leaves, we first showed that transient expression of bacterial feedback-resistant 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB) produced high titers of protocatechuate (PCA), which was in turn efficiently converted into PDC upon co-expression of PCA 4,5-dioxygenase (PmdAB) and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (PmdC) derived from Comamonas testosteroni. We validated that stable expression of AroG in Arabidopsis in a genetic background containing the QsuB gene enhanced PCA content in plant biomass, presumably via an increase of the carbon flux through the shikimate pathway. Further, introducing AroG and the PDC biosynthetic genes (PmdA, PmdB, and PmdC) into the Arabidopsis QsuB background, or introducing the five genes (AroG, QsuB, PmdA, PmdB, and PmdC) stacked on a single construct into wild-type plants, resulted in PDC titers of ~1% and ~3% dry weight in plant biomass, respectively. Consistent with previous studies of plants expressing QsuB, all PDC producing lines showed strong reduction in lignin content in stems. This low lignin trait was accompanied with improvements of biomass saccharification efficiency due to reduced cell wall recalcitrance to enzymatic degradation. Importantly, most transgenic lines showed no reduction in biomass yields. Therefore, we conclude that engineering plants with the proposed de-novo PDC pathway provides an avenue to enrich biomass with a value-added co-product while simultaneously improving biomass quality for the supply of fermentable sugars. Implementing this strategy into bioenergy crops has the potential to support existing microbial fermentation approaches that exploit lignocellulosic biomass feedstocks for PDC production., (Published by Elsevier Inc.)
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- 2021
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49. Draft Genome Sequence of Mycobacterium sp. Strain JC1 DSM 3803.
- Author
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Thompson MG, Eiben CB, Pearson AN, and Shih PM
- Abstract
Mycobacterium sp. strain JC1 DSM 3803 is one of the few known bacteria predicted to possess the xylulose monophosphate (XuMP) pathway of C
1 assimilation. The draft genome is 7,921,603 bp with a GC content of 66.88% and will allow more in-depth investigation of this bacterium's unique metabolism., (Copyright © 2021 Thompson et al.)- Published
- 2021
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50. Granick revisited: Synthesizing evolutionary and ecological evidence for the late origin of bacteriochlorophyll via ghost lineages and horizontal gene transfer.
- Author
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Ward LM and Shih PM
- Subjects
- Bacterial Proteins genetics, Bacteriochlorophylls genetics, Biological Evolution, Chlorophyll metabolism, Cyanobacteria genetics, Evolution, Molecular, Metabolic Networks and Pathways, Oxygen metabolism, Photosynthesis genetics, Photosynthesis physiology, Phylogeny, Bacteriochlorophylls metabolism, Gene Transfer, Horizontal genetics, Phototrophic Processes genetics
- Abstract
Photosynthesis-both oxygenic and more ancient anoxygenic forms-has fueled the bulk of primary productivity on Earth since it first evolved more than 3.4 billion years ago. However, the early evolutionary history of photosynthesis has been challenging to interpret due to the sparse, scattered distribution of metabolic pathways associated with photosynthesis, long timescales of evolution, and poor sampling of the true environmental diversity of photosynthetic bacteria. Here, we reconsider longstanding hypotheses for the evolutionary history of phototrophy by leveraging recent advances in metagenomic sequencing and phylogenetics to analyze relationships among phototrophic organisms and components of their photosynthesis pathways, including reaction centers and individual proteins and complexes involved in the multi-step synthesis of (bacterio)-chlorophyll pigments. We demonstrate that components of the photosynthetic apparatus have undergone extensive, independent histories of horizontal gene transfer. This suggests an evolutionary mode by which modular components of phototrophy are exchanged between diverse taxa in a piecemeal process that has led to biochemical innovation. We hypothesize that the evolution of extant anoxygenic photosynthetic bacteria has been spurred by ecological competition and restricted niches following the evolution of oxygenic Cyanobacteria and the accumulation of O2 in the atmosphere, leading to the relatively late evolution of bacteriochlorophyll pigments and the radiation of diverse crown group anoxygenic phototrophs. This hypothesis expands on the classic "Granick hypothesis" for the stepwise evolution of biochemical pathways, synthesizing recent expansion in our understanding of the diversity of phototrophic organisms as well as their evolving ecological context through Earth history., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
- Full Text
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